Abstract Heterotrimeric G proteins (???) are well known for their function in linking G protein-coupled receptors (GPCRs) to a variety of intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological processes. In the textbook view, G proteins carry out their function while associated with the cytoplasmic surface of a cell?s plasma membrane. In contrast to the classical view of plasma membrane-limited G protein signaling, it is becoming increasingly recognized that G protein localization is dynamic and regulated, such that they can reversibly traffic from the plasma membrane to intracellular locations, and that G proteins can have important cellular functions at intracellular sites. The research in this proposal focuses on understanding non-canonical functions of G?? subunits, and specifically roles for G?? in regulating signaling at the Golgi. Our previous work revealed an important role for Golgi-localized G?? in regulating a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the Golgi exit of select protein cargo destined for the plasma membrane. The research in this current proposal will examine the hypothesis that G?? regulates signaling pathways that control Golgi integrity by regulating the fragmentation of the Golgi under physiological and pathophysiological conditions. Reversible Golgi fragmentation is a cellular phenomenon that occurs under normal conditions, such as during mitosis, and that occurs in disease states, such as infection, cancer and neurodegenerative disease. This application will focus on defining a novel role for G?? in regulating the mitotic Golgi fragmentation checkpoint and by interrogating a novel role for G?? in regulating microtubule- dependent Golgi fragmentation. In addition, this application will define upstream mechanisms that directly promote signaling by G?? at the Golgi. These objectives will be pursued by a variety of experimental approaches, including cultured cells, immunofluorescence microscopy, fluorescence microscopy of live cells, biosensors, pharmacological inhibitors, mutational analysis, and biochemical assays.